Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.26.4 (RNase H)
 2,751 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The ability to selectively amplify RNA in the presence of genomic DNA of analogous sequence is cumbersome and requires implementation of critical controls for genes lacking introns. The convenient approaches of either designing oligonucleotide primers at the splice junction or differentiating the target sequence based on the size difference obtained by the presence of the intron are not possible. Our strategy for the selective amplification of RNA targets is based on the enzymology of a single thermostable DNA polymerase and the ability to modulate the strand separation temperature requirements for PCR amplification. Following reverse transcription of the RNA by recombinant Thermus thermophilus DNA polymerase (rTth pol), the resulting RNAxDNA hybrid is digested by the RNase H activity of rTth pol, allowing the PCR primer to hybridize and initiate second-strand cDNA synthesis. Substitution of one or more conventional nucleotides with nucleotide analogs that decrease base stacking interactions and/or hydrogen bonding (e.g. hydroxymethyldUTP or dITP) during the first- and second-strand cDNA synthesis step reduces the strand separation temperature of the resultant DNAxDNA duplex. Alteration of the thermal cycling parameters of the subsequent PCR amplification, such that the strand separation temperature is below that required for denaturation of genomic duplex DNA composed of standard nucleotides, prevents the genomic DNA from being denatured and therefore amplified.
Nucleic Acids Res 1996 Dec 15
PMID:Selective amplification of RNA utilizing the nucleotide analog dITP and Thermus thermophilus DNA polymerase. 901 75

Small nuclear RNAs (snRNA), cofactors in the splicing of pre-mRNA, are highly modified. In this report the modification of human U4 RNA was studied using cell extracts and in vitro synthesized, and therefore unmodified, U4 RNA. The formation of pseudouridine (Psi) at positions 4, 72 and 79 in U4 RNA was dependent on an RNA-containing cofactor, since the activities in the extracts were micrococcal nuclease (MN) sensitive. Extracts were fractionated on glycerol gradients and there was a broad peak of reconstitution activity centered at 14 S. Reconstitution was not due to additional enzymatic activity, since the peak fraction was MN sensitive. Oligodeoxynucleotide-mediated RNase H digestion of U6 RNA in the extracts inhibited formation of Psi in U4 RNA. From glycerol gradient analysis we determined that exogenously added U4 RNA that is associated with U6 RNA (sedimentation velocity 16 S) was significantly higher in Psi content than U4 RNA not associated with U6 RNA (8 S). Competitive inhibitors of Psi synthases, 5-fluorouridine-containing (5-FU) wild-type and mutant U4 RNAs, were used to investigate formation of Psi in U4 RNA. Deletions and point mutations in these 5-FU-containing U4 RNAs affected their ability to inhibit Psi synthase in vitro. With the aid of these potent inhibitors it was determined that at least two separate activities modify the uridines at these positions.
Nucleic Acids Res 1997 Dec 01
PMID:Modification of human U4 RNA requires U6 RNA and multiple pseudouridine synthases. 936 61

Hormone-sensitive lipase (HSL) catalyses the rate-limiting step of adipose tissue lipolysis. The human HSL gene is composed of nine exons encoding the adipocyte form and a testis-specific coding exon. Northern blot analyses showed that human adipocytes express a 2.8 kb HSL mRNA, suggesting the presence of a short (20-150 bp) 5' untranslated region (5'-UTR). A single 5'-UTR of approx. 70 nt was detected in RNase H mapping experiments. Two 5'-UTRs of 70 and 170 nt respectively were obtained by rapid amplification of cDNA ends and cDNA library screenings. RNase protection experiments, with probes derived from the two products, showed that human adipocyte HSL mRNA contains only the 70 nt product. Primer extension analysis mapped the transcriptional start site 74 nt upstream of the start codon. In HT29, a human cell line expressing HSL, the presence of the short or the long 5'-UTR is mutually exclusive. The short and long 5'-UTR exons were located 1.5 and approx. 13 kb respectively upstream of the first coding exon. Various portions of the 5'-flanking region upstream of the short product exon were linked to the luciferase gene and transfected into cells that express HSL (HT29 cells and rat adipocytes) and do not express HSL (HeLa cells). High luciferase activity was found for constructs containing the sequence between nt -2400 and -86, but not for shorter constructs. An analysis of 14 kb of genomic sequence revealed the presence of five DNase I hypersensitive sites associated with active gene transcription. Three of the sites are located in the vicinity of the transcriptional start site and could be linked to the minimal promoter activity. Two of the sites are located downstream of the exon containing the start codon, suggesting the presence of intronic regulatory elements.
Biochem J 1997 Dec 01
PMID:Characterization of the promoter of human adipocyte hormone-sensitive lipase. 937 1

A technique is described to identify the rare sequences within an RNA molecule that are available for efficient interaction with complementary DNA probes: the target RNA is digested by RNase H in the presence of a random pool of complementary DNA fragments generated from the same DNA preparation that was used for target RNA synthesis. The DNA region was amplified by PCR, partially digested with DNase and denatured prior to RNA binding. In the presence of single-stranded DNA fragments the RNA was digested with RNase H such that, on average, each molecule was cut once. Cleavage sites were detected by gel electrophoresis either directly with end-labeled RNA or by primer extension. The pattern of accessible sites on c- raf mRNA was determined and compared with the known profile of activity of oligonucleotides found in cells, showing the merit of the method for predicting oligonucleotides which are efficient for in vivo antisense targeting. New susceptible sites in the 3'-untranslated region of c- raf mRNA were identified. Also, four RNAs were probed to ascertain to what extent structure predicts accessibility: the P4-P6 domain of the Tetrahymena group I intron, yeast tRNAAsp, Escherichia coli tmRNA and a part of rat 18S rRNA.
Nucleic Acids Res 1997 Dec 15
PMID:A rapid in vitro method for obtaining RNA accessibility patterns for complementary DNA probes: correlation with an intracellular pattern and known RNA structures. 939 9

mRNA analogues-derivatives of oligoribonucleotides consisting of two different codons and bearing an aryl azide group at the 5'-phosphates-were crosslinked to human 80S ribosomes by UV-irradiation of the various model complexes obtained in the presence of the cognate tRNAs. Three sequences, namely pUUUGUU (coding for Phe and Val), pUUCUAAA (first triplet coding for Phe and second being stop-codon), and pGUGUUU (coding for Val and Phe), have been used. Sequences of 18S rRNA containing nucleotides crosslinked to the mRNA analogues were examined by hydrolysis with RNase H in the presence of various cDNA probes. Crosslinked nucleotides were identified by primer extension. In all cases, only nucleotide G-1207 (equivalent to G-926 in Escherichia coli 16S rRNA) has been detected as crosslinked. Crosslinking of the mRNA analogues to the large ribosomal subunit was negligible.
RNA 1997 Dec
PMID:Nucleotide G-1207 of 18S rRNA is an essential component of the human 80S ribosomal decoding center. 940 98

The circular DNA plasmid, designated pAAT56, has been isolated from strain T88-56 of the Japanese pear pathotype of Alternaria alternata. We determined the complete nucleotide sequence (5354 bp) of pAAT56 and mapped its possible open reading frames (ORFs). Three long ORFs, ORF1 (1290 bp), ORF2 (1653 bp) and ORF3 (690 bp), and four smaller ORFs, ORF4 to ORF7 (> or = 300 bp), were predicted from the sequence. The potential peptides derived from the ORFs other than ORF2 show no homology to other known proteins from a database search. However, ORF2 has significant homology to the pol gene of retrotransposons. The polypeptide derived from ORF2 includes sequences homologous to the reverse transcriptase (RT) and ribonuclease H (RNase H) domains of the retrotransposon Pol peptide. Phylogenetic comparison of RT domains from the retroelements placed pAAT56 in the Ty3/gypsy group of long terminal repeat (LTR) retrotransposons, most closely linked with those of filamentous fungi. The PCR primers were designed on the basis of nucleotide sequences encoding the highly-conserved amino-acid sequences in RT domains among pAAT56 and fungal retrotransposons. The PCR amplified the DNA fragments that possibly encode RT from strains of filamentous fungi that have been reported to carry retrotransposons. These results suggest that pAAT56 has acquired the pol gene from a Ty3/gypsy-group retrotransposon.
Gene 1997 Dec 05
PMID:Structural analysis of the plasmid pAAT56 of the filamentous fungus Alternaria alternata. 942 6

Sen1p from Saccharomyces cerevisiae is a nucleic acid helicase related to DEAD box RNA helicases and type I DNA helicases. The temperature-sensitive sen1-1 mutation located in the helicase motif alters the accumulation of pre-tRNAs, pre-rRNAs, and some small nuclear RNAs. In this report, we show that cells carrying sen1-1 exhibit altered accumulation of several small nucleolar RNAs (snoRNAs) immediately upon temperature shift. Using Northern blotting, RNase H cleavage, primer extension, and base compositional analysis, we detected three forms of the snoRNA snR13 in wild-type cells: an abundant TMG-capped 124-nucleotide (nt) mature form (snR13F) and two less abundant RNAs, including a heterogeneous population of approximately 1,400-nt 3'-extended forms (snR13R) and a 108-nt 5'-truncated form (snR13T) that is missing 16 nt at the 5' end. A subpopulation of snR13R contains the same 5' truncation. Newly synthesized snR13R RNA accumulates with time at the expense of snR13F following temperature shift of sen1-1 cells, suggesting a possible precursor-product relationship. snR13R and snR13T both increase in abundance at the restrictive temperature, indicating that Sen1p stabilizes the 5' end and promotes maturation of the 3' end. snR13F contains canonical C and D boxes common to many snoRNAs. The 5' end of snR13T and the 3' end of snR13F reside within C2U4 sequences that immediately flank the C and D boxes. A mutation in the 5' C2U4 repeat causes underaccumulation of snR13F, whereas mutations in the 3' C2U4 repeat cause the accumulation of two novel RNAs that migrate in the 500-nt range. At the restrictive temperature, double mutants carrying sen1-1 and mutations in the 3' C2U4 repeat show reduced accumulation of the novel RNAs and increased accumulation of snR13R RNA, indicating that Sen1p and the 3' C2U4 sequence act in a common pathway to facilitate 3' end formation. Based on these findings, we propose that Sen1p and the C2U4 repeats that flank the C and D boxes promote maturation of the 3' terminus and stability of the 5' terminus and are required for maximal rates of synthesis and levels of accumulation of mature snR13F.
Mol Cell Biol 1998 Dec
PMID:The putative nucleic acid helicase Sen1p is required for formation and stability of termini and for maximal rates of synthesis and levels of accumulation of small nucleolar RNAs in Saccharomyces cerevisiae. 981 77

The mechanism of reverse transcription was analyzed in vitro with RNA templates and the reverse transcriptase (RT) enzyme of human immunodeficiency virus type 1 (HIV-1). In particular, we analyzed the mechanism of actinomycin D (ActD) mediated inhibition of the strand transfer step, in which the newly synthesized cDNA, termed the (-) strand strong stop or (-)ssDNA, is transferred from the donor RNA onto the acceptor RNA. This strand transfer reaction is a rather inefficient process in vitro. We found that this is in part due to the presence of an excess donor RNA, and highly efficient strand transfer was achieved by reducing the amount of donor RNA. We suggest that annealing of the (-)ssDNA to the excess donor RNA is preferred over productive binding to the acceptor RNA because of a higher basepair complementarity. ActD remains a potent inhibitor of strand transfer in this optimized assay system. We measured no effect of ActD on the elongation of reverse transcription or the RNase H action of the RT enzyme. Instead, we provide evidence that ActD acts through direct interaction with the (-)ssDNA, thereby blocking the basepairing capacity of this molecule. The possible use of single-stranded DNA binding molecules as antiretroviral agents is discussed.
Nucleic Acids Res 1998 Dec 01
PMID:The mechanism of actinomycin D-mediated inhibition of HIV-1 reverse transcription. 982 74

Initiation of DNA plus-strand synthesis in most reverse-transcribing elements requires primer generation by reverse transcriptase-associated RNase H at one or more template polypurine tracts (PPTs). We have exploited infectious clones of the plant pararetrovirus cauliflower mosaic virus carrying redundant ectopic plus-strand priming elements to study priming in vivo. Ectopic priming generated an additional discontinuity in progeny virion DNA during infection of plants. We found that altering the length of the 13-base pair PPT by +/-25% significantly reduced priming efficiency. A short pyrimidine tract 5' to the PPT, highly conserved among diverse reverse-transcribing elements, was shown to play an important role in PPT recognition in vivo. The predominant DNA plus-strand 5' end remained 3 nucleotides from the PPT 3' end in mutant primers that were longer or shorter than the wild-type primer. Use of an ectopic redundant primer to study replication-dependent priming was validated by demonstrating that it could rescue infectivity following destruction of the wild-type priming elements. We propose a model for plant pararetroviral plus-strand priming in which pyrimidines enhance PPT recognition during polymerase-dependent RNase H cleavages, and suggest that fidelity of primer maturation during polymerase-independent cleavages involves PPT length measurement and 3' end recognition by RNase H.
J Biol Chem 1998 Dec 04
PMID:Analysis of polypurine tract-associated DNA plus-strand priming in vivo utilizing a plant pararetroviral vector carrying redundant ectopic priming elements. 982 93

We have used a ribonuclease protection assay to investigate RNase H cleavage of HIV-1 mRNA mediated by phosphorothioate antisense oligonucleotides complementary to the gag region of the HIV-1 genome in vitro. Cell lysate experiments in H9 and U937 cells chronically infected with HIV-1 IIIB showed RNase H cleavage of unspliced gag message but no cleavage of spliced message which did not contain the target gag region. RNase H cleavage products were detected at oligonucleotide concentrations as low as 0.01 microM and the RNase H activity was seen to be concentration dependent. Similar experiments with 1-, 3- and 5-mismatch oligonucleotides demonstrated sequence specificity at low concentrations, with cleavage of gag mRNA correlating with the predicted activities of the parent and mismatch oligonucleotides based on their hybridization melting temperatures. Experiments in living cells suggested that RNase H-specific antisense activity was largely determined by the amount of oligonucleotide taken up by the different cell lines studied. RNase H cleavage products were detected in antisense oligonucleotide treated MT-4 cells acutely infected with HIV-1 IIIB, but not in infected H9 cells treated with oligonucleotide under the same conditions. The data presented demonstrate potent and specific RNase H cleavage of HIV-1 mRNA mediated by an antisense oligonucleotide targeted to HIV-1 gag mRNA, and are in agreement with previous reports that the major obstacle to demonstrating antisense activity in living cells remains the lack of penetration of these agents into the desired cellular compartment.
Nucleic Acids Res 1998 Dec 15
PMID:Sequence-specific RNase H cleavage of gag mRNA from HIV-1 infected cells by an antisense oligonucleotide in vitro. 983 98


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